Multivalent Binding of Carbohydrates by the Human α-Defensin, HD5

Abstract

Four of the six human α-defensins (human neutrophil peptides 1–3 and human α-defensin 5; HD5) have a lectin-like ability to bind glycosylated proteins. Using HD5 as a model, we applied surface plasmon resonance techniques to gain insights into this property. HD5 bound natural glycoproteins > neoglycoproteins based on BSA > nonglycosylated BSA ≫ free sugars. The affinity of HD5 for simple sugars covalently bound to BSA was orders of magnitude greater than its affinity for the same sugars in solution. The affinity of HD5 for protein-bound carbohydrates resulted from multivalent interactions which may also involve noncarbohydrate residues of the proteins. HD5 showed concentration-dependent self-association that began at submicromolar concentrations and proceeded to dimer and tetramer formation at concentrations below 5 μM. The (R9A, R28A) and (R13A, R32A) analogs of HD5 showed greatly reduced self-association as well as minimal binding to BSA and to BSA-affixed sugars. From this and other evidence, we conclude that the extensive binding of HD5 to (neo)glycoproteins results from multivalent nonspecific interactions of individual HD5 molecules with carbohydrate and noncarbohydrate moieties of the target molecule and that the primary binding events are magnified and enhanced by subsequent in situ assembly and oligomerization of HD5. Self-association and multivalent binding may play integral roles in the ability of HD5 to protect against infections caused by viruses and other infectious agents.

Through the Looking Glass, Mechanistic Insights from Enantiomeric Human Defensins

“…Further, defensins can dimerize and potentially form higher order soluble aggregates in solution (6,55). It has been demonstrated that binding of HNP1 or HD5 to gp120 or bacterial toxins promotes defensin self-aggregation (56,82). Defensin oligomerization affords additional molecular complexity at the quaternary structural level, thereby contributing to enhanced structural diversity and functional versatility.…”

“…It is plausible that these surface electrostatic and hydrophobic forces, in combination with a disulfide-stabilized molecular scaffold, enable multiple defensin molecules to bind a single target protein molecule. In fact, both HNP1 and HD5 have been shown to bind bacterial toxins and HIV gp120 at a high molar ratio (56,82). Further, defensins can dimerize and potentially form higher order soluble aggregates in solution (6,55).…”

“…4, the disparity between L HNP1 and D HNP1 in their ability to bind HIV gp120 varied from 10-to 5-fold as the defensin concentration increased from 250 nM to 8 M. A similar disparity varying from 16-to 5-fold was found between L HD5 and D HD5 within the same concentration range. We did not try to fit the kinetic data to any mathematical model to obtain K d values due to known mechanistic complexities associated with multivalent carbohydrate-binding and binding-induced self-oligomerization of HNP1 and HD5 on the surface of glycoproteins (56).…”

Trp-26 Imparts Functional Versatility to Human α-Defensin HNP1

“…Trp-26 Mediates HNP1 Self-association-We have previously shown that ␣-defensins HNP1 and HD5 self-associate at high nM to low M concentrations, particularly in the presence of target proteins (34,62). To further examine the structural and functional role of Trp-26, we compared binding kinetics of HNP1, W26A-HNP1, and W26X-HNP1 on immobilized HNP1.…”

Functional Determinants of Human Enteric α-Defensin HD5

“…These small, 2-5-kDa peptides are classified into ␣, ␤, and families based on sequence homology and the connectivity of disulfide bonds linking the six conserved cysteine residues (6,7). Defensins bind carbohydrates (8,9), lipids (10,11), and DNA (12) and are active against a wide range of microorganisms, including bacteria (13,14) and viruses (15)(16)(17). Defensins are also capable of interacting with an diverse array of cellular receptors and host proteins, playing important immunomodulatory functions (5,18).…”